ENZYMES AS REAGENTS 



between lAA and enzyme-bound GSH, which was dependent 

 on the presence of DPN. By comparison, the interaction between 

 lAA and free GSH was quite slow. With three equivalents of 

 lAA, enzymatic activity of TDH was completely blocked within 

 a few minutes, and after proteolytic digestion of the modified 

 protein, no unreacted glutathione was liberated. Furthermore, 

 addition of acetyl phosphate to the enzyme led to the formation 

 of acyl enzyme, from which a thiol ester was liberated by 

 proteolytic digestion. Formation of acyl enzyme did not take 

 place after treatment with lAA, but if acetyl phosphate was 

 added prior to the inhibitor, the alkylation of the enzyme by 

 lAA was delayed. These findings with lAA and similar experi- 

 ments with A^-ethyl maleimide quite conclusively identified 

 GSH as part of the active center of TDH, and ruled out the 

 possibility that lAA inhibits enzymatic activity by steric hin- 

 drance of enzyme-substrate interaction. 



In the case of inhibitors of enzymes which act on large 

 molecular substrates, steric hindrance may, however, play an 

 important role. Some of the immunological antienzymes 

 probably act in this manner. The inhibition of trypsin by 

 ovomucoid inhibitor is dependent on groups which are non- 

 essential for proteolysis. This was shown by the fact that 

 acetylated trypsin, which is enzymatically active, is not readily 

 inhibited by ovomucoid inhibitor (9). It seems reasonable to 

 assume that steric hindrance plays a major role in the inhibition 

 of trypsin activity on proteins by ovomucoid, particularly since 

 both inhibitor and substrate are rather large molecular sub- 

 stances (2a). 



It is possible, on theoretical grounds, to differentiate three 

 types of chemical modifications which may result in altered 

 biological activity: (7) interaction with the active center; (2) 

 interaction with sites in the vicinity* of the active center leading 

 to steric hindrance (if the inhibitor is a large molecule, the site 



* In a coiled protein structure of the enzyme, a site in the vicinity of 

 the active center may be far removed in terms of the amino acid sequence of the 

 extended protein. 



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